Department of Molecular and Environmental Bioscience, Graduate School, Hanyang University, Seoul 133-791, South Korea.
Comp Biochem Physiol C Toxicol Pharmacol. 2011 Jun;154(1):19-27. doi: 10.1016/j.cbpc.2011.02.009. Epub 2011 Mar 3.
The monogonont rotifer, Brachionus sp. has been regarded as a potential model for reproductive physiology, evolution, and environmental genomics. To uncover the role of the heat shock protein upon temperature stress and hydrogen peroxide (H₂O₂) exposure, we cloned heat shock protein 20 (Hsp20) and determined its modulatory response under different temperatures and H₂O₂ concentrations. Under different temperature stresses (10 °C and 37 °C), the rotifer Brachionus sp. Hsp20 (Br-Hsp20) gene was highly expressed over time, and reached the maximum level 90 min after exposure, indicating that Br-Hsp20 gene would be involved in the chaperoning process to protect proteins at both low and high temperatures. To test the ability of thermotolerance of the recombinant Br-Hsp20-containing transformed Escherichia coli, we expressed the recombinant Br-Hsp20 protein with 1mM IPTG for 18 h at 30 °C, exposed them at 54 °C with time course (10 to 60 min), and measured cell survival. In this elevated temperature shock (54 °C), the cell survival was significantly higher at the Br-Hsp20 transformed E. coli, compared to the control (vector only). To analyze the modulatory effect of Br-Hsp20 gene on oxidative stress, we initially exposed 0.1 mM H₂O₂ over time and measured antioxidant enzyme activities along with the expression level of Br-Hsp20 mRNA. Upon H₂O₂ exposure, Br-Hsp20 gene was time-dependently upregulated and glutathione peroxidase (GPx), glutathione S-transferase (GST), and glutathione reductase (GR) activities were also elevated at the 12h-exposed group in a dose-dependent manner, indicating that the Br-Hsp20 gene would be an important gene in response to oxidative and temperature stress. Here, we demonstrated the role of the Hsp20 gene in the rotifer, Brachionus sp. providing a better understanding of the ecophysiology at environmental stress in this species.
单巢轮虫，Brachionus sp. 一直被认为是生殖生理学、进化和环境基因组学的潜在模式生物。为了揭示热休克蛋白在温度应激和过氧化氢 (H₂O₂) 暴露下的作用，我们克隆了热休克蛋白 20 (Hsp20)，并确定了其在不同温度和 H₂O₂浓度下的调节反应。在不同的温度应激（10°C 和 37°C）下，轮虫 Brachionus sp. Hsp20 (Br-Hsp20) 基因随时间呈高度表达，暴露后 90 分钟达到最高水平，表明 Br-Hsp20 基因将参与在低温和高温下对蛋白质的伴侣过程。为了测试重组含 Br-Hsp20 的大肠杆菌的耐热能力，我们用 1mM IPTG 在 30°C 下表达重组 Br-Hsp20 蛋白 18 小时，然后用时间过程（10 到 60 分钟）暴露在 54°C 下，并测量细胞存活率。在这种高温冲击（54°C）下，与对照（仅载体）相比，转化 Br-Hsp20 的大肠杆菌中的细胞存活率显著更高。为了分析 Br-Hsp20 基因对氧化应激的调节作用，我们首先随时间暴露于 0.1mM H₂O₂，并测量抗氧化酶活性以及 Br-Hsp20 mRNA 的表达水平。在 H₂O₂ 暴露下，Br-Hsp20 基因随时间呈上调趋势，谷胱甘肽过氧化物酶 (GPx)、谷胱甘肽 S-转移酶 (GST) 和谷胱甘肽还原酶 (GR) 活性也在 12 小时暴露组中呈剂量依赖性升高，表明 Br-Hsp20 基因将是对氧化和温度应激做出反应的重要基因。在这里，我们证明了 Hsp20 基因在轮虫 Brachionus sp. 中的作用，为该物种在环境应激下的生态生理学提供了更好的理解。
